POS0003 HUMAN LYMPH NODE STROMAL CELLS MAINTAIN FOXP3+ REGULATORY T CELLS IN A HLA-DR DEPENDENT MANNER
Authors: C. Grasso, A. M. O’byrne, J. F. Semmelink, E. Remmerswaal, J. W. Bolt, R. Mebius, L. Van Baarsen
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Animal studies have shown that lymph node stromal cells (LNSCs) are key players in peripheral tolerance through their capacity to present self-antigens in MHC class II to CD4+ T cells thereby controlling auto-reactivity and humoral response. Thus, it has been suggested that dysfunctional LNSCs may play a role in the breaking of tolerance observed in autoimmune diseases like rheumatoid arthritis (RA), where there is a genetic association with the HLA-DR locus. <h3>Objectives</h3> We hypothesise that human LNSCs from healthy individuals without autoimmunity can express HLA-DR and maintain FOXP3+ regulatory T cells, a process which may be disturbed in LNSCs from RA patients. <h3>Methods</h3> Lymph node needle biopsies were collected from healthy volunteers, autoantibody positive RA-risk individuals and RA patients. In addition, lymph node stromal cells (fibroblasts) and paired peripheral blood mononuclear cells were obtained from non-RA controls. A combination of imaging techniques including spectral analyser, ImageStream, confocal and electron microscopy was used to determine the expression of HLA-DR directly <i>ex vivo</i> and on <i>in vitro</i> cultured LNSCs. To investigate the ability of these LNSCs to maintain regulatory T cells we employed an <i>in vitro</i> system where LNSCs were co-cultured with autologous CD4+ T cells for 72 hours. Subsequently flow cytometry analysis was used to determine the presence of FOXP3+ regulatory T cells. <h3>Results</h3> The presence of lymphocytes induced HLA-DR expression on cultured LNSCs from non-RA controls. The presence of LNSCs within this autologous co-culture model is crucial for maintaining CD25+ CD127- FOXP3+ regulatory T cells. To investigate possible mechanisms for this maintenance we used blocking antibodies for both HLA-DR and interleukin 2 (IL-2) in our system and showed that the maintenance of FOXP3+ regulatory T cells required both HLA-DR and IL-2. In the context of autoimmunity we were able to show that lymph node biopsies from RA patients directly analysed <i>ex vivo</i> have a significant reduction in the frequency of HLA-DR+ LNSCs compared to control individuals and those at risk of developing the disease. <h3>Conclusion</h3> Overall, these results indicate an important role for HLA-DR expressing LNSCs in maintaining tolerance within the lymph node, a process that may be dysfunctional in the context of autoimmunity where tolerance is broken. <h3>Acknowledgements</h3> We thank our study subjects for participating in the study, the AMC radiology department for lymph node tissue sampling and the rheumatology lab for sample processing. The research leading to these results was funded by an AMC fellowship (to L.G.M.v.B.), the Dutch Arthritis Foundation LLP-30, a ZonMw TOP project 91217014 and the Dutch Organization for Health Research and Development (ZonMw) VIDI n° 91718371 (to L.G.M.v.B). <h3>Disclosure of Interests</h3> None Declared.